Synthesis, electrochemical properties, and antioxidant activity of sterically hindered catechols with 1,3,4-oxadiazole, 1,2,4-triazole, thiazole or pyridine fragments

A series of new RS−, RS−CH2− and R2N−CH2-functionalized сatechols with heterocyclic fragments such as 1,3,4-oxadiazole, 1,2,4-triazole, thiazole, or pyridine were synthesized by the reaction of 3,5-di-tert-butyl-o-benzoquinone or 3,5-di-tert-butyl-6-methoxymethylcatechol with different heterocyclic thiols. The S-functionalized catechols were prepared by the Michael reaction from 3,5-di-tert-butyl-o-benzoquinone and the corresponding thiols. The starting reagents such as substituted 1,3,4-oxadiazole-2-thiols and 4H-triazole-3-thiols are characterized by thiol–thione tautomerism, therefore their reactions with 3,5-di-tert-butyl-6-methoxymethylcatechol can proceed at the sulfur or nitrogen atom. In the case of mercapto-derivatives of thiazole or pyridine, this process leads to the formation of the corresponding thioethers with a methylene linker. At the same time, thiolated 1,3,4-oxadiazole or 1,2,4-triazole undergo alkylation at the nitrogen atom in the reaction with 3,5-di-tert-butyl-6-methoxymethylcatechol to form the corresponding thiones. The yield of reaction products ranges from 42 to 80%. The crystal structures of catechols with 3-nitropyridine or 1,3,4-oxadiazole-2(3H)-thione moieties were established by single-crystal X-ray analysis. The possibility of forming intra- and intermolecular hydrogen bonds has been established for these compounds. The electrochemical behavior of the studied compounds is influenced by several factors: the nature of the heterocycle and its substituents, the presence of a sulfur atom in the catechol ring, or a thione group in the heterocyclic core. The radical scavenging activity and antioxidant properties were determined using the reaction with synthetic radicals, the cupric reducing antioxidant capacity assay, the inhibition process of superoxide radical anion formation by xanthine oxidase, and the process of lipid peroxidation of rat liver (Wistar) homogenates in vitro.

chol ring, or a thione group in the heterocyclic core.The radical scavenging activity and antioxidant properties were determined using the reaction with synthetic radicals, the cupric reducing antioxidant capacity assay, the inhibition process of superoxide radical anion formation by xanthine oxidase, and the process of lipid peroxidation of rat liver (Wistar) homogenates in vitro.
Previously, we obtained a series of sterically hindered catechols linked through a sulfide bridge with various polar or lowpolar groups [36,[49][50][51] and heterocyclic fragments [52] via the Michael addition reaction.Also, we have previously found that the reaction between 3,5-di-tert-butyl-6-methoxymethylcatechol and different functionalized thiols occurs via an acid-catalyzed mechanism and leads to corresponding thioethers with methylene linker [53,54].Hybrid structures of this type are of particular interest from the point of view of studying their potential biological activity.This work aimed to obtain new multifunctional catechols containing pharmacologically active heterocyclic fragments such as thiazole, 1,3,4-oxadiazole, 1,2,4-triazole, and pyridine.We decided to use a non-hydrolyzable thioether group as a bridging moiety.A feature of heterocyclic thiols used as starting reagents is the possibility of thiol-thione tautomerism which leads to the appearance of two nucleophilic centers: a sulfur or nitrogen atom.Therefore, in the case of their interaction with 3,5-di-tertbutyl-6-methoxymethylcatechol, the possibility of alkylation of the nitrogen atom in the heterocycle cannot be excluded.This alternative pathway is similar to the previously described interaction of the aforementioned catechol with 3,5-dimethylpyrazole or benzimidazole [55].A study of the structure, electrochemical properties, anti/prooxidant, and antiradical activity was carried out for the catechols synthesized in this work.
The structures of synthesized compounds were confirmed by the spectral methods IR-, 1 H NMR, 13 C{ 1 H} NMR spectroscopy (Figures S1-S18 in Supporting Information File 1), HRMS, and elemental analysis.The molecular structures of compounds 5, 6 and 8 in crystal state were determined by single-crystal X-ray analysis.

X-ray data
The X-ray suitable crystals 5, 6•0.5CH 3 CN, and 8 were grown by slow recrystallization of the compounds from acetonitrile solutions.The unit cells of complexes 5 and 8 do not contain any solvent molecules, while the unit cell of crystals 6•0.5CH 3 CN contains one acetonitrile molecule per two catechol molecules.The nitro-substituted pyridine group in 5 is disordered in two positions.The X-ray structure of catechol 5 is shown in Figure 1, and the X-ray structures of catechols 6, and 8 are shown in Figure 2.
At the same time, for molecule B, there is a hydrogen bond between the O2B-H2B group with the nitrogen atom N3 of the  The intermolecular H-bonding O2-H2•••O1 between catechol moieties of two neighbouring molecules leading to pairs was also observed in the crystal of catechol 8 (Figure 5): The H2•••O1 distance is 2.06 (1) Å, the O2•••O1 distance is 2.87(1) Å, the angle O2-H2-O1 is 151.4°.However, the additional intermolecular contacts O1-H1 1) Å, the angle O1-H1-N3 is 168.3°) with the pyridine group of the catechol moiety of the neighbouring pair lead to the formation of chains.As a result of such interaction, the hydroxy group O1-H1 is turned away from the neighbouring group O2-H2, and there is no intramolecular hydrogen bond between hydroxy groups in molecule 8 (Figure 2(b)).A π-stalking was found between the pyridine groups in molecules belonging to the neighbouring pairs with the corresponding distance of 3.45(1) Å.
In the anodic area, the electrochemical profile for catechol thioethers 1-5 represents two successive irreversible oxidative stages.The first two-electron peak is observed in the range of 0.94-1.25 V.It refers to the oxidation of the catechol moiety to the corresponding o-benzoquinone, as previously shown for related compounds [36].The second redox transition at 1.55-1.84V characterizes the oxidation of the sulfide fragment (Scheme 2).The replacement of the oxadiazole cycle with a thiazole one in catechol thioether 3 leads to a shift in the oxidation peak of the catechol fragment to the cathode region (Figure 7).This is due to the less acceptor nature of the thiazole ring compared to the more electron-deficient oxadiazole cycle, which contains three heteroatoms.In addition, according to NMR spectral data, one proton of the hydroxy group of catechol 3 is shifted to the thiazole nitrogen atom, which also affects the decrease in the oxidation potential.In the case of 3, the second anodic peak corresponding to thioether oxidation is multielectronic and shifted towards more positive values.More intensive current can be explained by the proximity of the thioether linker and the thiazole moiety oxidation potentials.
The appearance of a methylene bridge between the aromatic ring and the sulfur atom in catechol 4 leads to a shift of the catechol oxidation peak to the cathodic area (Supporting Information File 1, Figure S20) as compared to catechol 3.This effect is due to a decrease in the acceptor effect of the thioether group.
In turn, the second two-electron stage for 4 is recorded at earlier potential values.On the contrary, this behavior is caused by the presence of CH 2 -linker which divides electrogenerated quinone form and heterocycle moiety.The formation of intramolecular hydrogen bonds between the OH group and the pyridine ring facilitates the electrooxidation process (1.,0 V) (Supporting Information File 1, Figure S21) for catechol thioether 5.The second oxidation peak is shifted to the anodic region due to the influence of the electron-withdrawing nitro group.Some differences are observed in the electrochemical behavior of thiones 6-9 compared to thioethers 1-5.In the case of compounds 6-8 with a 1,3,4-oxadiazole-2-thione fragment, the oxidation potential of the first peak is shifted to the cathodic region compared to catechols 1 and 2 which contain a similar heterocycle (Figure 8 for 7, Supporting Information File 1, Figures S22 and S23 group in catechols 1 and 2 with a similar heterocyclic fragment (Figure 6).The presence of an acceptor 3-pyridyl ring in the 1,3,4-oxadiazole-2-thione core for compound 8, leads to a shift of E pa2 by 0.22-0.26V to the anodic region compared to compounds 6 and 7.In addition, the current intensity of the second peak corresponding to the transfer of two electrons increases (Figure S23 in Supporting Information File 1).The electrochemical behavior of catechol 9 with a 1,2,4-triazole-5-thione ring was studied in a MeCN-DMSO mixture to increase its solubility.The first redox transition is detected at early potential values (0.85 V) which coincides with the value obtained for the corresponding starting thiol.However, there is a peak at E = −0.11V on the reverse CV branch of 9, characteristic of compounds with a catechol fragment (Figure S24 in Supporting Information File 1).The second anodic peak for 9 is not observed due to the narrow electrochemical window of the solvent in the positive CV region.The formation of o-benzoquinones at the first stage of oxidation was confirmed during a controlled potential microelectrolysis of compound 6 at 1.30 V (2 h, 0.8 F/mol).A similar electrochemical picture is observed for electrolysis products of catechol 6 as in the case of catechol 3. The recorded absorption spectra after the electrolysis display an intense maximum at λ = 412 nm which is characteristic for sterically hindered o-benzoquinones.The proposed scheme for the electrooxidation of catechols 6-9 is presented in Scheme 3.
Based on the current intensities of the second anodic peaks for compounds 6-8 with a 1,3,4-oxadiazole-2-thione ring (Figure 8 for 7, Supporting Information File 1, Figures S22 and S23 for 6 and 8, respectively), it can be assumed that the second redox transition can occur in one (for 6 and 7) and a two-electron (for 8) manner.
Thus, the electrochemical behavior of the studied catechols with heterocyclic fragments is influenced by several factors: the nature of the heterocycle and its substituents, the presence/ absence of a sulfur atom in the catechol ring or thione sulfur atom in the heterocyclic core.Based on electrochemical data, it can be assumed that the most pronounced antioxidant activity can be exhibited by compounds with minimal first oxidation potentials: catechol 4 containing a 4-phenylthiazole ring and catechol thione 9 with a 1,2,4-triazole ring.

Radical scavenging, anti/prooxidant activity of catechols with heterocyclic fragments
The radical scavenging properties and antioxidant activity of catechols 1-9 were investigated in the reaction with a diphenylpicrylhydrazyl (DPPH) radical, ABTS •+ radical cation, CUPRAC test, and inhibition process of superoxide radical anion formation by xanthine oxidase (NBT assay).The presence of a catechol fragment and thioether or thione groups determines the ability to neutralize different radical species.A comparative evaluation of the antioxidant activity of catechols 1-9 and Trolox was performed in a reaction with DPPH in dichloromethane at 298 K (Table 2).
Based on the IC 50 value, compound 3 with a 4-phenylthiazole ring linked through a sulfur atom to the catechol ring exhibits the highest activity (13.0 ± 1.0 µM) in the reaction with the DPPH radical.The number of converted DPPH molecules per one molecule of 3 is equal to two.Comparable IC 50 values were obtained for catechol thioethers 4 and 5 with methylene bridge.
The use of ABTS radical cation to assess the antioxidant capacity of compounds is one of the widely used methods which is based on the transfer of an electron from the studied molecules to the acceptor [67].We have previously shown that catechol thioethers can act as anti-or prooxidants on lipid peroxidation processes in vitro depending on a functional group or a heterocycle substitute at the sulfur atom [49,50,54].Non-enzymatic process of the rat (Wistar) liver homogenate lipid peroxidation (LP) in the presence of additives of target compounds 1-9 was studied in vitro.
The concentration of the carbonyl compounds forming in the reaction of the lipid peroxidation was determined by the accumulation of a colored complex with thiobarbituric acid (TBARS) (Figure 9).
A decrease in TBARS concentration was observed in the presence of all tested compounds which indicates their antioxidant effect during incubation time.At the initial stage (3 h), catechol thione 8 containing a pyridine substituent in the 1,3,4-oxadiazole-2-thione cycle has the greatest inhibitory effect (36%) on the LP process compared to the control sample.For compounds 2, 4-7, and 9, a less pronounced antioxidant effect (13-19%) is observed comparable to the inhibitory activity of Trolox.Initially (3 h), catechols 1 and 3 exhibit a weak inhibitory effect (7-8%).An increase in the incubation time to 24 hours leads to a rise in antioxidant activity for all compounds.A decrease in TBARS content by 41-52% was noted compared to the control experiment.All the studied compounds exhibit more effective inhibitory activity than Trolox.The inhibitory effect for compounds 2, 5, 6, 8, and 9 increases to 47-64% after 48 hours.This indicates the ability of these catechols to have a prolonged effect on the LP process.In the presence of Trolox, amounts of TBARS decrease by 33% over 48 hours of incubation.
The C TBARS value varies in a narrow range of values and is practically independent of experimental time for 5 and 6.This behavior indicates the occurrence of an induction period during lipid peroxidation when the level of TBARS remains constant.
In the case of 1 and 7, a decrease in the inhibitory effect to 17-28% is observed with an increase in incubation time from 24 to 48 hours.Catechol thioether 5 with a 3-nitro-substituted 2-pyridine moiety and 8 with a 3-pyridine substituent in the 1,3,4-oxadiazole-2-thione ring demonstrate the maximum antioxidant activity (63-64%) over 48 hours of the experiment.A comparative assessment of the effect of thioethers 1 and 2 in the LP process points out that the replacement of the methyl group in the phenyl ring with a chlorine atom leads to increased antioxidant activity of 2. In the presence of catechol thioether 3, the TBARS content is slightly lower compared to 4. A phenyl substitute in the thiazole ring in 4 affects negatively its antioxi- dant activity in the LP process compared to CatH 2 -CH 2 -S-thiazolyl reducing the inhibitory effect from 81% to 43% (48 h).Consequently, catechol thioethers 2 and 5 as well as catechol thione 8 are the most effective antioxidants in the lipid peroxidation reaction.

Conclusion
Thus, we have synthesized a series of new sterically hindered catechols with thioether or thione groups containing various heterocyclic fragments with good yields of 42-80%.The interaction of 3,5-di-tert-butyl-o-benzoquinone with heterocyclic thiols leads to the formation of S-functionalized catechols.In the case of the acid-catalyzed reaction between 3,5-di-tertbutyl-6-methoxymethylcatechol and mercapto-substituted thiazole or pyridine derivatives, catechol−thioethers are formed with an additional methylene bridge between the catechol ring and the sulfur atom.In contrast, the mercapto derivatives of 1,3,4-oxadiazole or 1,2,4-triazole undergo alkylation at the nitrogen atom in the reaction with 3,5-di-tert-butyl-6methoxymethylcatechol which leads to the corresponding catechol thiones.The molecular structures of catechols with a 2-pyridine or 1,3,4-oxadiazole-2-thione ring in the crystalline state were studied by X-ray diffraction.
It has been established that the number of heteroatoms in the cycle which are bonded to the catechol ring through a sulfur atom and/or methylene bridge as well as the presence of thioether or thione groups affect the electrochemical behavior of catechols.The electrochemical data made it possible to determine compounds characterized by minimal anodic potentials (4 and 9) which may exhibit more pronounced antioxidant activity among the studied compounds.
Catechol thioethers 3 and 4 proved to be the most effective scavengers of the DPPH radical.Thiones 6, 7, and 9 showed pronounced antiradical activity in the reaction with ABTS •+ .Compounds 3, 4, and 8 showed high inhibitory activity in NBTtest.All studied compounds have antioxidant effects on the process of lipid peroxidation.Thus, in most of the studied assays, compounds 3 and 4 with a 4-phenylthiazole moiety turned out to be effective antioxidants among catechol thioethers, and 8 and 9 among thiones with a catechol fragment, which is consistent with electrochemical data.

Figure 1 :
Figure 1: The X-ray structure of catechol 5 (the thermal ellipsoids of 50% probability).The hydrogen atoms except those of hydroxy groups are omitted.

Figure 2 :
Figure 2: The X-ray structures of catechols 6 (a) and 8 (b) (the thermal ellipsoids of 50% probability).The hydrogen atoms except those of hydroxy groups are omitted.

Figure 3 :
Figure 3: Fragment of the pack of catechol 5 in crystal (the H-bonds and π-π interactions are shown as dotted lines).

Figure 4 :
Figure 4: The interactions in pair of independent molecules A and B of 6 in crystal 6•0.5CH 3 CN (the H-bonds are shown as dotted lines).

Figure 5 :
Figure 5: Fragment of the pack of catechol 8 in crystal (the H-bonds and π-π interactions are shown as dotted lines).

Table 1 :
The peak potentials of 1-9 obtained by the CV (GC-electrode, CH 3 CN, ν = 0.2 V•s −1 , 0.1 М n-Bu 4 NClO 4 , c = 1-3 mmol•L −1 To confirm the participation of the catechol group in the first redox transition, the microelectrolysis of 3 was carried out at a controlled potential of 1.35 V in MeCN (2 h, 0.8 F/mol).After electrolysis, a decrease in the current intensity of the first oxidation peak is observed on the CVs of this compound (conversion reaches 51%).Besides, the current value of the second peak remains unchanged.In the cathodic region, a one-electron quasi-reversible peak is displayed (E pc = −0.58V).This process corresponds to the reduction of o-benzoquinone to o-benzosemiquinone.During electrolysis, the color of the solution changes.This is confirmed by the appearance of an absorption maximum in the UV-visible spectrum at λ = 490 nm, which is characteristic of thiolated o-benzoquinones.This absorption band corresponds to intramolecular charge transfer between the thioether group and the o-benzoquinone fragment[36,[62][63][64][65].The potentials of the first anodic peak of thioethers 1 and 2 with an oxadiazole core are fixed at close values.(Supporting Information File 1, FigureS19for 1, Figure6for 2).

Figure 9 :
Figure 9: The level of TBARS in rat liver homogenates in vitro, in the presence of compounds 1-9, Trolox, and without additives (control) during the incubation for 3, 24, and 48 hours (concentration of compounds in the reaction medium is 0.1 mM).The results are expressed as mean ± standard deviation (* p < 0.001; ** p < 0.01; ***p < 0.05).
The obtained IC 50 values for synthesized catechols vary over a wide range from 10.5 to 45.6 μM.As in the case of the reaction with the DPPH radical, thioethers 1 and 2 possess a maximal IC 50 value which indicates their low radical scavenging activity.Compounds 3 and 4 containing a 4-phenylthiazole moiety have similar IC 50 values.This points out that the methylene bridge separating the catechol ring and the sulfur atom does not influence their neutralizing activity.The presence of a phenyl substituent in the heterocyclic fragment of compound 4 has a positive effect on the antiradical activity in this test in comparison with CatH 2 -CH 2 -Sthiazolyl.The electron-withdrawing NO 2 group in the pyridine ring of catechol 5 suppresses its antiradical properties in comparison with CatH 2 -CH 2 -S-pyridinyl which is similar to the patterns observed in the DPPH test.In general, compounds with thione moieties were more active ABTS •+ scavengers than thioethers 1-5 except catechol 8.The minimum IC 50 values in this test were obtained for compounds 6 and 7. Therefore, catechol thiones 6, 7, and 9 were more effective ABTS •+ scavengers than Trolox. in which the sulfur atom is directly bonded to the aromatic ring.The IC 50 value is 63.48 ± 1.08 μM for compound 4. The appearance of a phenyl substituent in the thiazole ring affects negatively the antiradical ability of compound 4 com-pared to its unsubstituted analogue CatH 2 -CH 2 -S-thiazolyl (34.0 ± 0.10 μM).Compound 1 was the least effective in this test (81.10 ± 1.86 μM) as in the case of the methods described above.